Most tissues in the body, such as skin, liver and peripheral nerve, have a remarkable ability to repair themselves after injury. By contrast, the central nervous system (CNS)—including the brain and the spinal cord—has little innate capacity for repair. When axonal connections are damaged in the adult brain or spinal cord, they show an extremely limited ability to regenerate, even though axons can grow and regenerate efficiently in the embryonic CNS and in the adult peripheral nervous system. Factors that account for the inability of CNS axons to regenerate can be grouped in two categories: intrinsic properties of CNS neurons that may make them incapable of regeneration; and extrinsic factors in the CNS environment that are inhibitory to axonal elongation.
The idea that factors in the CNS environment can prevent regeneration dates back to the early 20th century. Ramon y Cajal observed that the inability of adult CNS neurons to extend axonal processes could be overcome by giving them the permissive environment of a peripheral nerve. Then, about 20 years ago, David and Aguayo showed that retinal neurons could form long projections in peripheral nerve grafts. Later, Schwab discovered that dorsal root ganglion neurons in culture extend their axons across Schwann cells but avoid oligodendrocytes and the fatty myelin sheath (Schwab et al 1993).
These results show that failure to regenerate is not purely an intrinsic deficit of CNS neurons, and inhibitory factors in the CNS environment also play an important role. These inhibitory factors are mainly located in the glial scar that forms at the region of injury and by the myelin that ensheaths axons in the white matter tracks.
Following CNS injury, the central area of necrosis is infiltrated by glia and other non-neuronal cells, and a fibrous scar forms. Axons do not extend through the scar and their growth appears to be inhibited by it. Molecular components that may contribute to this inhibitory activity include the extracellular matrix glycoprotein tenascin-R (TN-R) and the myelin-associated neurite outgrowth inhibitors myelin-associated glycoprotein (MAG) and Nogo.
TN-R
TN-R has been implicated in a variety of cell-matrix interactions involved in the molecular control of axon guidance and neural cell migration during development and regeneration of CNS (reviewed by Erickson, 1993; Chiquet-Ehrismann et al., 1994; Pesheva et al., 2000; 2001). TN-R is the smallest member of the tenascin family and is composed of four structural motifs: a cysteine-rich segment at the N-terminus is followed by 4.5 EGF-like repeats. This region is followed by 9 consecutive fibronectin type III-like domains and at the C-terminus TN-R is related to the beta- and- gamma-chains of fibrinogen.
TN-R is expressed predominantly by oligodendrocytes during the onset and early phases of myelin formation, and remains expressed by some oligodendrocytes in the adult. TN-R is also expressed in some neurons and interneurons in the spinal cord, retina, cerebellum, and hippocampus (Fuss et al., 1991; 1993). TN-R co-localizes with other glial-derived molecules (i.e. myelin-associated glycoprotein and a phosphacan-related molecule) at high density in the nodes of Ranvier of CNS myelinated axons (Xiao et al., 1997: Yang et al, 1999).
TN-R can inhibit or promote neurite outgrowth, depending on the neuronal cell type and the environment in which it is presented. When TN-R was offered, acting as a sharp substrate boundary, dorsal root ganglion (DRG), cerebellum and retinal ganglion neuron growth cones avoided growing on these molecules, but were not induced to collapse. On the other hand, when TN-R was offered in a mixture with laminin (which strongly promotes growth of embryonic and adult axons) as a uniform substrate, DRG growth cones displayed a collapsed morphology and were able to advance at a faster rate than on laminin alone.
Using several monoclonal antibodies binding to distinct epitopes on the tenascin molecule, epidermal growth factor-like (EGF-L) repeats and fibronectin type III homologous repeats 4-5 were identified to be responsible for growth cone repulsion.
In vitro, outgrowth of embryonic and adult retinal ganglion cell axons from mouse retinal explants is significantly reduced on homogeneous substrates of tenascin-R or a bacterially expressed tenascin-R fragment comprising the EGF-L domain. When both molecules are presented, acting as a sharp substrate boundary, regrowing adult axons do not cross into the territory containing tenascin-R or EGF-L. All in vitro experiments were done in the presence of laminin, suggesting that tenascin-R and EGF-L actively inhibit axonal growth. Neurites and growth cones were repelled from areas coated with fragments containing the EGF-L (the amino-terminal cysteine-rich domain plus the EGF-like repeats), FN (fibronectin) 1-2, FN3-5 and FG (fibrinogen) domains of TN-R, and EGF-L prevents neurite outgrowth of hippocampal neurons.
TN-R also induces axonal defasciculation in vitro through the EGF-L domain (Taylor et al., 1993; Xiao et al., 1996, 1997, 1998; Becker et al, 1999; Becker et al, 2000).
After 3-acetylpyridine-induced lesion of the olivocerebellar system of the adult rat, the density of cells containing TN-R transcripts increased significantly in the inferior olivary nucleus and in the white matter of the cerebellar cortex. Immunohistochemical investigations confirmed these observations at the protein level.
After a spinal cord mechanical lesion of rat, TN-R mRNA was also upregulated (Wintergerst at al, 1997; Deckner et al, 2000).
These findings suggested that the continued overexpression of TN-R in the injured CNS may contribute to the failure of adult axonal regeneration in vivo.
Tenascin-R is a member of the tenascin family, which play important roles in cell interactions in the developing nervous system, such as neuronal migration, neuritogenesis, and neuronal regeneration. Tenascin-R is expressed predominantly by oligodendrocytes during the onset and early phases of myelin formation, and remain expressed by some oligodendrocytes in the adult (Pesheva et. al., 1989; Fuss et al., 1991,1993; Wintergerst et. al., 1993; Ajemian A. et. al., 1994). TN-R is a multi-functional molecule (Lochter and Schachner, 1993; Pesheva et. al., 1993; Taylor et. al., 1993; Xiao et. al., 1996, 1997,1998), and it has indicated that it is an inhibitory component of myelin extraction. Xiao have found that, the EGF-L domain of TN-R can inhibit the neurite outgrowth.
MAG
MAG is a transmembrane protein of the immunoglobulin superfamily expressed by myelinating glial cells of the central and peripheral nervous systems, where MAG represents 1 and 0.1% of the total myelin proteins, respectively (Heape at al, 1999). MAG is a potent inhibitor of axonal regeneration and also, depending on the age and type of neuron, can promote axonal growth. MAG inhibits neurite outgrowth of retinal, superior cervical ganglion, spinal, and hippocampal and dorsal root ganglion (DRG) neurons of all postnatal ages, but can enhance neurite outgrowth of embryonic spinal cord neurons and newborn DRG neurons (DeBellard at al, 1996; Turnley et al, 1998; Shen et al, 1998; Yang et al, 1999).
MAG can also induce growth cone collapse. 60% of axonal growth cones of postnatal day 1 hippocampal neurons collapsed when they encountered coated recombinant MAG (rMAG). Such collapse was not observed with denatured rMAG (Li et al, 1996). Soluble dMAG (a proteolytic fragment of the extracellular domain of MAG, which is released in abundance from myelin and found in vivo) and chimeric MAG-Fc can potently inhibit neurite outgrowth from P6 DRG neurons. This inhibition was blocked when a MAG monoclonal antibody was included.
These results indicate that soluble dMAG detected in vivo could contribute to the lack of regeneration in the mammalian CNS after injury (Tang et al, 1997; 2001).
MAG has two recognition sites for neurons, the sialic acid binding site at R118 and a distinct inhibition site which is absent from the first three Ig domains (Tang et al, 1997).
MAG is a well characterized member of the immunoglobulin gene superfamily, and it exerts a robust inhibitory effect on neurite outgrowth from young cerebellar neurons and adult dorsal ganglion (DRG) neurons (Mukhopadhyay et al., 1994). MAG is a membrane protein with 626 amino acids. It has been reported that soluble MAG, which consists of the extracellular domain, has an inhibitory effect on neurite outgrowth (Mckerracher et. al., 1994). The extracellular domain of MAG consists of five Ig-like domains, and it is demonstrated that the first two Ig-like domains are important for the interaction between MAG and neuronal membrane, while the other three Ig-like domains might be involved in the inhibitory effects (Collins et al., 1997).
Nogo
Nogo is a high molecular weight integral membrane protein that localizes to CNS myelin, but not PNS myelin. Nogo has three isoforms, named Nogo-A, -B and -C, which are generated by alternative splicing. NI-250 and NI-35 were first identified and named as the 2 isoforms of Nogo; it has now been established that NI-250 is Nogo-A and NI-35 is Nogo-B. Nogo is expressed by oligodendrocytes in white matter of the CNS and is found in the inner and outer leaflets of myelin and in the endoplasmic reticulum.
In vitro characterization of Nogo has demonstrated its function as a potent inhibitor of axon elongation. In vivo neutralization of Nogo activity results in enhanced axonal regeneration and functional recovery following CNS injury as well as increased plasticity in uninjured CNS fibers. The monoclonal antibody mAb IN-1 was shown to promote long-distance regeneration and functional recovery in vivo when applied to spinal cord-injured adult rat (Chen et al, 2000; GrandPre et al; Merkler et al).
These findings suggest that Nogo may be a major contributor to the nonpermissive nature of the CNS environment. Two distinct inhibitory domains of Nogo have been identified: an intracellular amino-terminal domain (NogoN) of Nogo A and a short 66 residue region (Nogo-66) located between two hydrophobic domains of the three isoforms, Nogo-A, Nogo-B and Nogo-C (Chen et al, 2000; GrandPre et al, 2000; Fournier et al , 2001; Filbin, 2003). These domains are illustrated in Science, Vol 297 (5584), 16 Aug. 2002, p. 1132-1134.
Nogo-A is expressed by oligodendrocytes but not by Schwann cells. It can inhibit axonal extension and collapses dorsal root ganglion growth cones (GrandPre et. al., 2000). The neurite outgrowth inhibitory activity of Nogo can be neutralized by monoclonal antibody IN-1, which allows axonal regeneration and functional recovery after spinal cord injury (Chen et. al., 2000). Nogo is a membrane protein with 1163 amino acids. The C-terminal tail contains two hydrophobic transmembrane domains separated by a 66-residue hydrophilic extracellular domain. This 66-residue extracellular domain can inhibit axon outgrowth (Fournier et. al., 2001).
Huang et al. (1999) discloses a therapeutic vaccine approach to stimulate axonal regeneration in the adult spinal cord.
It is an object of the invention to provide materials and methods useful in the treatment of, or in the development of treatments for, CNS damage, e.g. spinal cord injury, by overcoming the inhibitory effects of myelin on axonal regeneration.